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. 2012 Jul 18;7(7):e41185. doi: 10.1371/journal.pone.0041185

Figure 6. Rem2 and CaMKII redistribution is co-dependent.

Figure 6

(A) CaMKIIN inhibits redistribution of Rem2 and CaMKII. Rat hippocampal neurons coexpressing (left) GFP-Rem2 and (right) mRuby-CaMKII without (left panels) and with (right panels) HA-CaMKIIN. Cells were imaged before stimulation (top panels), then stimulated with 100 µM glutamate/10 µM glycine and imaged again (middle panels). Neurons were fixed and probed for HA to detect CaMKIIN (bottom panel). The scale bar represents 20 µm. (B) CaMKIIN reduces redistribution of coexpressed CaMKII and Rem2. Clustering factor determined as in [2]. Mean ± SEM clustering factor for CaMKII without CaMKIIN: before stimulation, 0.004±0.002; after, 0.021±0.004; with CaMKIIN: before, 0.002±0.0002; after, 0.009±0.002. Rem2 clustering without CaMKIIN: before stimulation, 0.001±0.0002; after, 0.009±0.002; with CaMKIIN: before, 0.001±0.0002; after, 0.003±0.0006. Data is from 4 separate experiments with a total of N = 27 neurons/condition. Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test). (C) Rem2 and CaMKII redistributions overlap temporally. A timecourse of aggregation of Rem2 and CaMKII shows little clustering before stimulation with glutamate/glycine, and Rem2 and CaMKII clustering occurs at similar rates. N = 27 neurons/condition. (D) CaMKIIN does not interact with Rem2, and expression of CaMKIIN does not interfere with Rem2-CaMKII interaction. HEK cells were transfected with the indicated plasmids. Cell lysates were subjected to co-precipitation assays using a mix of proteinA/G beads and anti-HA,-myc or-GFP. Eluates were separated on SDS-PAGE and probed with anti-HA (top) and anti-myc (bottom). (E) CaMKII aggregates in HEK cells following a pH drop/high Ca protocol [2] (mean clustering factor before simulation 0.030±0.002; after 0.166±0.013). Aggregation is inhibited by CaMKIIN (before stimulation, 0.024±0.002; after, 0.068±0.010) but unaffected by coexpression of Rem2 (before, 0.031±0.003; after, 0.152±0.009). CaMKIIN inhibition is also insensitive to Rem2 coexpression (before, 0.031±0.003; after, 0.071±0.006). N = 20 cells and p<0.05 for all before-after pairs. (F) Rem2 does not aggregate using the same protocol (without CaMKIIN: before stim, 0.037±0.004; after, 0.032±0.004; with CaMKIIN: before, 0.018±0.002; after, 0.022±0.003), unless CaMKII is also present (without CaMKIIN: before stim, 0.012±0.002; after, 0.073±0.008). CaMKII-induced Rem2 aggregation is also inhibited by CaMKIIN (before, 0.008±0.001; after, 0.021±0.004). Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test).